Genetic markers associated with age-related macular degeneration, methods of detection and uses thereof

ABSTRACT

Disclosed is a method for identifying an individual who has an altered risk for developing age related macular degeneration comprising detecting a single nucleotide polymorphism (SNP)

This application is a divisional of Ser. No. 12/382,569 (allowed), filedMar. 18, 2009 (published as US 2009-0269761-A1), which claims thebenefit of U.S. Provisional Application No. 61/037,411 filed on Mar. 18,2008, the entire contents of each of which is hereby incorporated byreference.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of genetic testing, drugdiscovery, and Age-Related Macular Degeneration. In particular, itrelates to genetic variants found within the complement cascade C3 genewhich increase the risk of Age-Related Macular Degeneration.

BACKGROUND OF THE INVENTION

Age-related macular degeneration (AMD) causes progressive impairment ofcentral vision and is the leading cause of irreversible vision loss inolder Americans(1). The most severe form of AMD involvesneovascular/exudative (wet) and/or atrophic (dry) changes to the macula.Although the etiology of AMD remains largely unknown, implicated riskfactors include age, ethnicity, smoking, hypertension, obesity and diet(2). Familial aggregation (3), twin studies (4), and segregationanalysis(5) suggest that there is also a significant geneticcontribution to the disease. The candidate gene approach and genome-wideassociation studies have consistently implicated the CFH, ARMS2 andC2/BF genes, all members of the complement-mediated inflammatorycascade.

Age-related macular degeneration (AMD) is a common complex disorder thataffects the central region of the retina (macula) and is the leadingcause of legal blindness in older American adults. The prevalence of AMDand its significant morbidity will rise sharply as the population ages.AMD is a clinically heterogeneous disorder with a poorly understoodetiology. Population-based longitudinal studies(6-8) have establishedthat the presence of extracellular protein/lipid deposits (drusen)between the basal lamina of the retinal pigment epithelium (RPE) and theinner layer of Bruchs' membrane is associated with an increased risk ofprogressing to an advanced form of AMD, either geographic atrophy orexudative disease. The presence of large and indistinct (soft) drusencoupled with RPE abnormalities is considered an early form of thedisorder and is often referred to as age-related maculopathy (ARM).

Epidemiology: AMD is a complex disorder with contributions ofenvironmental factors as well as genetic susceptibility(2). Manyenvironmental and lifestyle factors have been postulated, but by far themost consistently implicated non-genetic risk factor for AMD iscigarette smoking (6). Much progress has been made in identifying andcharacterizing the genetic basis of AMD. In a remarkable example of theconvergence of methods for disease gene discovery, multiple independentresearch efforts identified the Y402H variant in the complement factor H(CFH [(MIM 134370]) gene on chromosome 1q32 as the first major AMDsusceptibility allele (9-14). While one of the studies was able topinpoint CFH on the basis of a whole-genome association study (11), moststudies focused on the 1q32 region because it had consistently beenimplicated by several whole-genome linkage scans. Disease associatedhaplotypes within the CFH gene are also associated with AMD (15). Asecond genomic region with similarly consistent linkage evidence ischromosome 10q26, which was identified as the single most promisingregion by a recent meta-analysis of published linkage screens (16).

Two studies have suggested specific AMD susceptibility genes located onchromosome 10q26. One used a combination of family-based andcase-control analyses to implicate the PLEKHA1 gene (pleckstrin homologydomain containing, family A (phosphoinositide binding specific) member 1[MIM 607772]) and the predicted ARMS2 gene (14;17;18). ARMS2 appears tobe a mitochondrial membrane protein involved in inflammation (19) Asecond study using two independent case-control datasets concluded thatthe T allele of SNP rs10490924 in ARMS2, a coding change (Ala69Ser) inexon 1 of this gene, was the most likely AMD susceptibility allele (16).Both studies reported that the chromosome 10q26 variant confers an AMDrisk similar in magnitude to that of the Y402H variant in CFH. A locuswith less strong association, but reproducible association with AMD isthe complement component 2 (C2) and Factor B (C2/BF) locus within themajor histocompatability complex III locus found on chromosome 6 The L9Hvariant of BF and the E318D variant of C2 , as well as a variant inintron 10 of C2 and the R32Q variant of BF, confer a significantlyreduced risk of AMD (20).

SUMMARY OF THE INVENTION

Here, we describe highly significant association of SNPs within the C3gene (NCBI GeneID: 718), specifically rs2230199 (Arg102Gly) found onchromosome 19 with age related macular degeneration and its use, aloneor in combination, in predicting predisposition to this disease (21). Wehave thus established that identification of the nucleotide residue atrs2230199 can predict the predisposition of an individual to AMD.Related findings have since been published by Maller et al. (22).

According to some embodiments of the invention, a method is provided forassessing increased risk of Age Related Macular Degeneration. Theidentity is determined of at least one nucleotide residue of the genomicgerm-line C3 coding sequence of an individual The nucleotide residue isidentified as normal or variant by comparing it to a normal genomicgerm-line sequence of C3 coding sequence as shown in SEQ ID NO:1 (codingsequence) or SEQ ID NO: 3 (genomic sequence). A normal nucleotideresidue is identical to the corresponding nucleotide residue in thenormal genomic germ-line sequence of C3. A variant nucleotide residue isnot identical to the corresponding nucleotide residue in the normalgenomic germ-line sequence of C3. A variant C3 coding sequence maycontain at least one variant nucleotide residue relative to the normalC3 coding sequence. An individual with a variant sequence has a higherrisk of Age Related Macular Degeneration than an individual with anormal sequence.

According to some embodiments, a method is provided for assessingincreased risk of Age Related Macular Degeneration. The identity isdetermined of at least one amino acid residue of the C3 protein of anindividual. The at least one amino acid residue is identified as normalor variant by comparing it to a normal sequence of the C3 protein asshown in SEQ ID NO: 2. A person with a variant sequence has a higherrisk of Age Related Macular Degeneration than a person with a normalsequence.

Further embodiments of the invention provide a method to assess risk ofAMD in an individual. The presence of a G or C allele at the singlenucleotide polymorphism (SNP)rs 2230199 within the genomic sequence isdetermined in an individual. The person is identified as being at highrisk of AMD if the patient has one or two copies of the G allele on thenegative genomic strand at this SNP (or conversely one or two copies ofthe C allele on the positive genomic strand) in relation to the March2006 human reference sequence (NCBI Build 36.1). . The SNP rs2230199 isfound in the first position of codon 102 (corresponding to position 366in the C3 coding sequence of SEQ ID NO: 1 or 304 nucleotides downstreamof the start of the initiation codon). SNP rs2230199 is located atposition 6669387 on human chromosome 19 ((NCBI Build 36.1). The G allelechanges the amino acid specified from arginine to glycine. The patientis identified as being at lower risk of AMD if the patient does not haveone or two copies of the G allele at rs2230199.

Further embodiments provide a method for assessing increased risk of AgeRelated Macular Degeneration. The identity of the residue at position102 of the pro-C3 protein sequence or position 80 of the mature C3protein sequence is determined in an individual. The residue isidentified as normal or variant by comparing it to a normal sequence ofthe pro-C3 protein or C3 protein as shown in SEQ ID NO: 2. An individualwith a variant sequence has a higher risk of Age Related MacularDegeneration than an individual with a normal sequence. For example, anindividual with Gly at position 102 has a higher risk of Age RelatedMacular Degeneration than an individual with Arg at position 102.

While not being bound by any theory, this marker, or one in linkagedisequilibrium, may change the composition, function or abundance of theelements of cellular constituents resulting in a predisposition to agerelated macular degeneration. Measuring this marker in individuals whodo not ostensibly have age related macular degeneration may identifythose at heightened risk for the subsequent development of age relatedmacular degeneration, providing benefit for, but not limited to,individuals, insurers, care givers and employers. Information obtainedfrom the detection of SNPs associated with age related maculardegeneration is of great value in the treatment and prevention of thiscondition.

In the context of this invention, a marker is said to be in “linkagedisequilibrium” with the residue at rs2230199 when the correlationcoefficient (r²) between the marker and rs2230199 is >0.5 (23).

Further scope of the applicability of the present invention will becomeapparent from the detailed description provided below. It should beunderstood however, that the following detailed description andexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodification within the spirit and scope of the invention will becomeapparent to those skilled in the art from the following

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have discovered that polymorphic variants in theC3 gene, which is shown in sequences, SEQ ID NOs: 1-3 are associatedwith an altered risk of developing age related macular degeneration insubjects. The present invention thus provides a SNP associated with agerelated macular degeneration, nucleic acid molecules containing the SNP,methods and reagents for the detection of the SNP disclosed herein, usesof this SNP for the development of detection reagents, and assays orkits that utilize such reagents. The age related maculardegeneration-associated SNP disclosed herein may be useful fordiagnosing, screening for, and evaluating predisposition to age relatedmacular degeneration and related pathologies in humans.

The age related macular degeneration-associated SNP has been identifiedby genotyping DNA from 1548 individuals, 847 of these individuals havingbeen previously diagnosed with age related macular degeneration and 701being “control” or individuals thought to be free of age related maculardegeneration.

Aspects of the present invention thus provides an individual SNPassociated with age related macular degeneration, genomic sequences (SEQID NO: 3) containing SNPs, transcript sequences (SEQ ID NO: 1) and aminoacid sequences (SEQ ID NO: 2). Aspects of the invention include methodsof detecting these polymorphisms in a test sample, methods ofdetermining the risk of an individual of having or developing agerelated macular degeneration, methods of using the disclosed SNPs toselect a treatment strategy, and methods of using the SNPs of thepresent invention for human identification.

When the presence in the genome of an individual of a particular base,e.g., guanine, at a particular location in the genome (e.g. the SNPrs2230199) correlates with an increased probability of that individualcontracting age related macular degeneration vis-à-vis a population nothaving that base at that location in the genome, that individual is saidto be at “increased risk” of contracting age related maculardegeneration , i.e., to have an increased susceptibility. In the presentcase, such increased probability exists when the base is present in oneor the other or both alleles of the individual. Furthemore, theprobability is increased when the base is present in both alleles of theindividual rather than one allele of the individual.

When the presence in the genome of an individual of a particular base,e.g., cytosine, at a particular location in the genome (e.g. the SNPrs2230199) decreases the probability of that individual contracting agerelated macular degeneration vis-à-vis a population not having that baseat that location in the genome, that individual is said to be at“decreased risk” of contracting age related macular degeneration, i.e.,to have a decreased susceptibility. Such an allele is sometimes referredto in the art as being “protective”. As with increased risk, it is alsopossible for a decreased risk to be characterized as dominant orrecessive.

An “altered risk” means either an increased or a decreased risk.

The genetic analysis detailed below-linked age related maculardegeneration with a SNP in the human genome. A SNP is a particular typeof polymorphic site, a polymorphic site being a region in a nucleic acidsequence at which two or more alternative nucleotides are observed in asignificant number of individuals from a population. A polymorphic sitemay be a nucleotide sequence of two or more nucleotides, an insertednucleotide or nucleotide sequence, a deleted nucleotide or nucleotidesequence, or a microsatellite, for example. A polymorphic site that istwo or more nucleotides in length may be 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or more, 20 or more, 30 or more, 50 or more, 75 or more,100 or more, 500 or more, or about 1000 nucleotides in length, where allor some of the nucleotide sequences differ within the region. Thespecific polymorphic site found in the genomic sequences identified asSEQ ID NOs: 1 and 3 is a “single nucleotide polymorphism” or a “SNP”i.e. a polymorphic site which is one nucleotide in length.

Where there are two, three, or four alternative nucleotide sequences ata polymorphic site, each nucleotide sequence is referred to as a“polymorphic variant” or “nucleic acid variant.” Where two polymorphicvariants exist, for example, the polymorphic variant represented in amajority of samples from a population is sometimes referred to as a“prevalent allele” and the polymorphic variant that is less prevalentlyrepresented is sometimes referred to as an “uncommon allele.” Anindividual who possesses two prevalent alleles or two uncommon allelesis “homozygous” with respect to the polymorphism, and an individual whopossesses one prevalent allele and one uncommon allele is “heterozygous”with respect to the polymorphism. Individuals who are homozygous withrespect to one allele are sometimes predisposed to a different phenotypeas compared to individuals who are heterozygous or homozygous withrespect to another allele.

A genotype or polymorphic variant may also be expressed in terms of a“haplotype,” which refers to the identity of two or more polymorphicvariants occurring within genomic DNA on the same strand of DNA. Forexample, two SNPs may exist within a gene where each SNP position mayinclude a cytosine variation or an adenine variation. Certainindividuals in a population may carry an allele (heterozygous) or twoalleles (homozygous) having the gene with a cytosine at each SNPposition. As the two cytosines corresponding to each SNP in the genetravel together on one or both alleles in these individuals, theindividuals can be characterized as having a cytosine/cytosine haplotypewith respect to the two SNPs in the gene.

A “phenotype” is a trait which can be compared between individuals, suchas presence or absence of a condition, for example, occurrence of agerelated macular degeneration.

Polymorphic variants are often reported without any determination ofwhether the variant is represented in a significant fraction of apopulation. Some reported variants are sequencing errors and/or notbiologically relevant. Thus, it is often not known whether a reportedpolymorphic variant is statistically significant or biologicallyrelevant until the presence of the variant is detected in a populationof individuals and the frequency of the variant is determined.

A polymorphic variant may be detected on either or both strands of adouble-stranded nucleic acid. Also, a polymorphic variant may be locatedwithin an intron or exon of a gene or within a portion of a regulatoryregion such as a promoter, a 5′ untranslated region (UTR), a 3′ UTR, andin DNA (e.g., genomic DNA (gDNA) and complementary DNA (cDNA)), RNA(e.g., mRNA, tRNA, and rRNA), or a polypeptide. Polymorphic variationsmay or may not result in detectable differences in gene expression,polypeptide structure, or polypeptide function.

In our genetic analysis associating age related macular degenerationwith the polymorphic variants set forth in Table 1, samples fromindividuals diagnosed with age related macular degeneration andindividuals not having age related macular degeneration were allelotypedand genotyped. The allele frequency for each polymorphic variant amongcases and controls was determined. These allele frequencies werecompared in cases and controls, or combinations. Particular SNPs werethus found to be associated with age related macular degeneration whengenotype and haplotype frequency differences calculated between case andcontrol pools were established to be statistically significant.

As mentioned above, polymorphic variants can travel together. Suchvariants are said to be in “linkage disequilibrium” so that heritableelements e.g., alleles that have a tendency to be inherited togetherinstead of being inherited independently by random assortment are inlinkage disequilibrium. Alleles are randomly assorted or inheritedindependently of each other if the frequency of the two alleles togetheris the product of the frequencies of the two alleles individually. Forexample, if two alleles at different polymorphic sites are present in50% of the chromosomes in a population, then they would be said toassort randomly if the two alleles are present together on 25% of thechromosomes in the population. A higher percentage would mean that thetwo alleles are linked. For example, a first polymorphic site P1 havingtwo alleles, e.g. A and C--each appearing in 50% of the individuals in agiven population, is said to be in linkage disequilibrium with a secondpolymorphic site P2 having two alleles e.g. G and T—each appearing in50% of the individuals in a given population, if particular combinationsof alleles are observed in individuals at a frequency greater than 25%(if the polymorphic sites are not linked, then one would expect a 50%chance of an individual having A at P1 and a 50% chance of having G atP2 thus leading to a 25% chance of having the combination of A at P1 andG at P2 together). Heritable elements that are in linkage disequilibriumare said to be “linked” or “genetically linked” to each other.

One can see that in the case of a group of SNPs that are in linkagedisequilibrium with each other, knowledge of the existence of all suchSNPs in a particular individual generally provides redundantinformation. Thus, when identifying an individual who has an alteredrisk for developing age related macular degeneration according to thisinvention, it is necessary to detect only one SNP of such a group ofSNPs associated with an altered risk of developing age related maculardegeneration.

The data set out below shows that one or more SNPs in the C3 genomicsequences identified herein as SEQ ID NOs: 1 and 3 are associated withthe occurrence of age related macular degeneration. Thus, featuredherein are methods for identifying a risk of age related maculardegeneration in a subject, which includes detecting the presence orabsence of a polymorphic variant at one or more of the SNPs describedherein in a human nucleic acid sample. For example, the presence orabsence of a polymorphic variant at rs2230199 (e.g. the G allele) may bedetected in a human nucleic acid sample.

Three different analyses were performed for each marker and significantresults reported below as follows: (a) a test of trend across the 3genotypes(24) , (b) a dominant model where the homozygous genotype forallele “B” is combined with the prevalent heterozygote genotype; and (c)a recessive model where the homozygous genotype for allele “A” iscombined with the heterozygous genotype. An empirical p-value for thelargest of these three test statistics was calculated by permutations.In addition, a Mantel-Haenszel odds ratio measuring the change in riskassociated with each additional copy of allele B is also calculated andreported.

Pertinent results for the SNP are summarized in Table 1: Chromosomalnumber and position-using the International Human Genome SequencingConsortium build 35 (http://www.ncbi.nlm.nih.gov/genome/seq/) as madeavailable by the National Center for Biotechnology Information (NCBI),National Library of Medicine, Building 38A, Bethesda, Md. 20894 U.S.A.,gene marker name-using the nomenclature of the NCBI dbSNP(URL[colon][slash][slash]www[dot]ncbi[dot]nlm[dot]nih[dot]gov[slash]SNP[slash]) and genename-using the unigene naming convention. Under the “Case Flag” thenumber 1 designates Cases and the number 0 designates Controls. Theidentity of the base designated “A” in the analysis is indicated where1=A (adenine), 2=C (cytosine), 3=G (guanine) and 4=T (thymidine). “B”indicates the polymorphic allele. AA, AB, BB are the counts of thenumber of individuals with the given genotype, by cases/controls. Theodds ratio is the Mantel-Haenszel odds ratio across the three genotypes.

It has been discovered that polymorphic variation at SNPs in the C3genomic sequences which are identified herein as SEQ ID NOs: 1 or 3 isassociated with the occurrence of age related macular degeneration.Thus, featured herein are methods for identifying a risk of age relatedmacular degeneration in a subject, which comprises detecting thepresence or absence of one or more of the polymorphic variationsdescribed herein in a human nucleic acid sample. The polymorphicvariations and SNPs are detailed in the table. In some embodiments, thepresence of a polymorphic variant at rs2230199 is indicative of analtered risk of age related macular degeneration. For example, thepresence of the uncommon G allele at rs2230199 may be indicative of anincreased risk of age related macular degeneration, relative toindividuals with the prevalent C allele at rs2230199.

Methods for determining whether a subject is at risk of age relatedmacular degeneration are provided herein. These methods includedetecting the presence or absence of one or more polymorphic variationsat SNPs which are associated with age related macular degeneration, in asample from a subject.

SNPs may be associated with a disease state such as AMD, in humans or inanimals. The association can be direct, as in conditions where thesubstitution of a base results in alteration of the protein codingsequence of a gene which contributes directly to the pathophysiology ofthe condition. Common examples of this include diseases such as sicklecell anemia and cystic fibrosis. The association can be indirect whenthe SNP plays no role in the disease, but is located close to thedefective gene such that there is a strong association between thepresence of the SNP and the disease state. Because of the high frequencyof SNPs within the genome, there is a greater probability that a SNPwill be linked to a genetic locus of interest than other types ofgenetic markers.

Disease-associated SNPs may occur in coding and non-coding regions ofthe genome. When located in the coding region altered function of theensuing protein sequence may occur. For example, polymorphic variationat SNP rs2230199 may alter the amino acid residue at position 102 of theC3 pro-protein. If it occurs in the regulatory region of a gene it mayaffect expression of the protein. Ifthe protein is involved inprotecting the body against pathological conditions this can result indisease susceptibility.

Numerous methods exist for the measurement of specific SNP genotypes.Individuals carrying mutations in one or more SNPs of the presentinvention may be detected at the DNA level by a variety of techniques.Nucleic acids for diagnosis may be obtained from a patient's cells, suchas from blood, urine, saliva, tissue biopsy and autopsy material.

The genomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR prior to analysis (25). RNA or cDNA may alsobe used in the same ways. As an example, PCR primers complementary tothe nucleic acid of one or more SNPs of the present invention can beused to identify and analyze the presence or absence of the SNP. Forexample, deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Pointmutations can be identified by hybridizing amplified DNA to radiolabeledSNP RNA of the present invention or alternatively, radiolabeled SNPantisense DNA sequences of the present invention. Perfectly matchedsequences can be distinguished from mismatched duplexes by RNase Adigestion or by differences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures(26).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method(27).

Thus, the detection of a specific DNA sequence may be achieved bymethods which include, but are not limited to, hybridization, RNaseprotection, chemical cleavage, direct DNA sequencing or the use ofrestriction enzymes, (e.g., restriction fragment length polymorphisms(“RFLP”) and Southern blotting of genomic DNA).

Hybridisation may be carried out under stringent hybridizationconditions, for example for detection of sequences that are about 80-90%identical suitable conditions include hybridization overnight at 42° C.in 0.25M Na₂HPO₄, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final washat 55° C. in 0.1×SSC, 0.1% SDS. For detection of sequences that aregreater than about 90% identical, suitable conditions includehybridization overnight at 65° C. in 0.25M Na₂HPO₄, pH 7.2, 6.5% SDS,10% dextran sulfate and a final wash at 60° C. in 0.1×SSC, 0.1% SDS.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

Genetic mutations can be identified by hybridizing a sample and controlnucleic acids, e.g., DNA or RNA, to high density arrays containinghundreds or thousands of oligonucleotides probes(28;29). For example,genetic mutations can be identified in two-dimensional arrays containinglight-generated DNA probes as described in Cronin et al., supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

Specific mutations can also be determined through direct sequencing ofone or both strands of DNA using dideoxy nucleotide chain terminationchemistry, electrophoresis through a semi-solid matrix and fluorescentor radioactive chain length detection techniques. Further mutationdetection techniques may involve differential susceptibility of thepolymorphic double strand to restriction endonuclease digestion, oraltered electrophoretic gel mobility of single or double stranded genefragments containing one polymorphic form. Other techniques to detectspecific DNA polymorphisms or mutation may involve evaluation of thestructural characteristics at the site of polymorphism using nuclearmagnetic resonance or x-ray diffraction techniques.

These genetic tests are useful for prognosing and/or diagnosing agerelated macular degeneration and often are useful for determiningwhether an individual is at an increased or decreased risk of developingor having age related macular degeneration .

Thus, the invention includes a method for identifying a subject at riskof age related macular degeneration , which includes detecting in anucleic acid sample from the subject the presence or absence of apolymorphic variant at a SNP associated with age related maculardegeneration in a nucleotide sequence identified as SEQ ID NOs:1 and 3.

For example, the presence of one or two copies of the G allele at SNPrs2230199 may be indicative of the subject being at risk of age relatedmacular degeneration i.e. an individual at risk of AMD may beheterozygous (genotype GC) or homozygous (genotype GG) at SNP rs2230199in the C3 gene,

Results from prognostic tests may be combined with other test results todiagnose age related macular degeneration. For example, prognosticresults may be gathered, a patient sample may be ordered based on adetermined predisposition to age related macular degeneration , thepatient sample analyzed, and the results of the analysis may be utilizedto diagnose age related macular degeneration . Also age related maculardegeneration diagnostic methods can be developed from studies used togenerate prognostic/diagnostic methods in which populations arestratified into subpopulations having different progressions of agerelated macular degeneration . In some embodiments, prognostic resultsmay be gathered; a patient's risk factors for developing age relatedmacular degeneration analyzed (e.g., age, family history, smoking); anda patient sample may be ordered based on a determined predisposition toage related macular degeneration. In some embodiments, the results frompredisposition analyses may be combined with other test results,epidemiologic or genetic in nature, indicative of age related maculardegeneration, which were previously, concurrently, or subsequentlygathered with respect to the predisposition testing. In theseembodiments, the combination of the prognostic test results with othertest results can be probative of age related macular degeneration, andthe combination can be utilized as a age related macular degenerationdiagnostic.

Risk of age related macular degeneration sometimes is expressed as aprobability, such as an odds ratio, percentage, or risk factor. The riskis based upon the presence or absence of the SNP variant describedherein, and also may be based in part upon phenotypic traits of theindividual being tested. Methods for calculating risk based upon patientdata are well known (30). Allelotyping and genotyping analyses may becarried out in populations other than those exemplified herein toenhance the predictive power of the prognostic method. These furtheranalyses are executed in view of the exemplified procedures describedherein, and may be based upon the same polymorphic variations oradditional polymorphic variations. Risk determinations for age relatedmacular degeneration are useful in a variety of applications. In someembodiments, age related macular degeneration risk determinations may beused by clinicians to direct appropriate detection, preventative andtreatment procedures to subjects who most require these. In otherembodiments, age related macular degeneration risk determinations may beused by health insurers for preparing actuarial tables and forcalculating insurance premiums.

The nucleic acid sample typically is isolated from a biological sampleobtained from a subject. For example, nucleic acid can be isolated fromblood, saliva, sputum, urine, cell scrapings, and biopsy tissue. Thenucleic acid sample can be isolated from a biological sample usingstandard techniques. The nucleic acid sample may be isolated from thesubject and then directly utilized in a method for determining thepresence of a polymorphic variant, or alternatively, the sample may beisolated and then stored (e.g., frozen) for a period of time beforebeing subjected to analysis.

The presence or absence of a polymorphic variant may be determined usingone or both chromosomal complements represented in the nucleic acidsample. Determining the presence or absence of a polymorphic variant inboth chromosomal complements represented in a nucleic acid sample isuseful for determining the zygosity of an individual for the polymorphicvariant (i.e., whether the individual is homozygous or heterozygous forthe polymorphic variant). For example, a homozygous individual havingthe GG genotype at SNP rs2230199 (i.e. the G allele in both copies ofthe C3 gene) may have an increased risk of AMD relative to aheterozygous individual having the GC genotype at SNP rs2230199 (i.e.the G allele in one copies of the C3 gene and the C allele in the other)

Any oligonucleotide-based diagnostic may be utilized to determinewhether a sample includes the presence or absence of a polymorphicvariant in a sample. For example, primer extension methods, ligasesequence determination methods (e.g., U.S. Pat. Nos. 5,679,524 and5,952,174, and WO 01/27326), mismatch sequence determination methods(e.g., U.S. Pat. Nos. 5,851,770; 5,958,692; 6,110,684; and 6,183,958),microarray sequence determination methods, restriction fragment lengthpolymorphism (RFLP), single strand conformation polymorphism detection(SSCP) (e.g., U.S. Pat. Nos. 5,891,625 and 6,013,499), PCR-based assays(e.g., TAQMAN™ PCR System (Applied Biosystems)), and nucleotidesequencing methods may be used.

Oligonucleotide extension methods typically involve providing a pair ofoligonucleotide primers in a polymerase chain reaction (PCR) or in othernucleic acid amplification methods for the purpose of amplifying aregion from the nucleic acid sample that comprises the polymorphicvariation. One oligonucleotide primer is complementary to a region 3′ ofthe polymorphism and the other is complementary to a region 5′ of thepolymorphism. A PCR primer pair may be used in methods disclosed in U.S.Pat. Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143;6,140,054; WO 01/27327; and WO 01/27329 for example. PCR primer pairsmay also be used in any commercially available machines that performPCR, such as any of the GENEAMPTM, systems available from AppliedBiosystems. Also, those of ordinary skill in the art will be able todesign oligonucleotide primers based upon the nucleotide sequences setforth in SEQ ID NOs: 1 and 3.

Also provided is an extension oligonucleotide that hybridizes to theamplified fragment adjacent to the polymorphic variation. An adjacentfragment refers to the 3′ end of the extension oligonucleotide beingoften 1 nucleotide from the 5′ end of the polymorphic site, andsometimes 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5′ end ofthe polymorphic site, in the nucleic acid when the extensionoligonucleotide is hybridized to the nucleic acid. The extensionoligonucleotide then is extended by one or more nucleotides, and thenumber and/or type of nucleotides that are added to the extensionoligonucleotide determine whether the polymorphic variant is present.Oligonucleotide extension methods are disclosed, for example, in U.S.Pat. Nos. 4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934;5,908,755; 5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431;6,017,702; 6,046,005; 6,087,095; 6,210,891; and WO 01/20039.Oligonucleotide extension methods using mass spectrometry are described,for example, in U.S. Pat. Nos. 5,547,835; 5,605,798; 5,691,141;5,849,542; 5,869,242; 5,928,906; 6,043,031; and 6,194,144. Multipleextension oligonucleotides may be utilized in one reaction, which isreferred to as multiplexing.

A microarray can be utilized for determining whether a SNP is present orabsent in a nucleic acid sample. A microarray may include anyoligonucleotides described herein, and methods for making and usingoligonucleotide microarrays suitable for diagnostic use are disclosed inU.S. Pat. Nos. 5,492,806; 5,525,464; 5,589,330; 5,695,940; 5,849,483;6,018,041; 6,045,996; 6,136,541; 6,142,681; 6,156;501; 6,197,506;6,223,127; 6,225,625; 6,229,911; 6,239,273; WO 00/52625; WO 01/25485;and WO 01/29259. The microarray typically comprises a solid support andthe oligonucleotides may be linked to this solid support by covalentbonds or by non-covalent interactions. The oligonucleotides may also belinked to the solid support directly or by a spacer molecule. Amicroarray may comprise one or more oligonucleotides complementary to anucleotide sequence which includes a SNP set forth in Table 1. The oneor more oligonucleotides may for example, hybridise specifically to anucleotide sequence which comprises a particular polymorphic variant atthe SNP, but not to nucleotide sequences which comprise otherpolymorphic variants at the SNP.A kit also may be utilized fordetermining whether a polymorphic variant is present or absent in anucleic acid sample. A kit may include one or more pairs ofoligonucleotide primers useful for amplifying a fragment of a nucleotidesequence of interest, where the fragment includes a polymorphic site.The kit may comprise a polymerizing agent, for example, a thermostablenucleic acid polymerase such as one disclosed in U.S. Pat. Nos.4,889,818 or 6,077,664. Also, the kit may comprise an elongationoligonucleotide that hybridizes to the nucleotide sequence in a nucleicacid sample adjacent to the polymorphic site. Where the kit includes anelongation oligonucleotide, it may also comprise chain elongatingnucleotides, such as dATP, dTTP, dGTP, dCTP, and dITP, including analogsof dATP, dTTP, dGTP, dCTP and dITP, provided that such analogs aresubstrates for a thermostable nucleic acid polymerase and can beincorporated into a nucleic acid chain elongated from the extensionoligonucleotide. Along with chain elongating nucleotides may be one ormore chain terminating nucleotides such as ddATP, ddTTP, ddGTP, ddCTP.The kit may comprise one or more oligonucleotide primer pairs, apolymerizing agent, chain elongating nucleotides, at least oneelongation oligonucleotide, and one or more chain terminatingnucleotides. Kits optionally include buffers, vials, microtiter plates,and instructions for use.

An individual identified as being susceptible to age related maculardegeneration may be heterozygous or homozygous with respect to theallele associated with an increased risk of age related maculardegeneration, as indicated in the table. For example, the individual maybe heterozygous or homozygous with respect to the G allele of rs2230199which is shown herein to be associated with an increased risk of agerelated macular degeneration. A subject homozygous for an alleleassociated with an increased risk of age related macular degeneration isat a comparatively high risk of age related macular degeneration as faras that SNP is concerned whether or not the allelic effect has beendetermined to be dominant or recessive. A subject who is heterozygousfor an allele associated with an increased risk of age related maculardegeneration , in which the allelic effect is recessive would likely beat a comparatively reduced risk of age related macular degenerationpredicted by that SNP. The allelic effect of the G allele of rs2230199is shown herein to be dominant and an individual who is heterozygous forthe G allele may be at an increased risk of age related maculardegeneration relative to individuals who lack the G allele.

Individuals carrying mutations in one or more SNP of the presentinvention may be detected at the protein level by a variety oftechniques. Cells suitable for diagnosis may be obtained from apatient's blood, urine, saliva, tissue biopsy and autopsy material.

Also featured are methods for determining risk of age related maculardegeneration and/or identifying a subject at risk of age related maculardegeneration by contacting a polypeptide or protein encoded by anucleotide sequence from a subject with an antibody that specificallybinds to an epitope associated with an altered, usually increased riskof age related macular degeneration in the polypeptide.

Another aspect of the invention provides an isolated nucleic acidmolecule comprising at least 8, or at least 9, or at least 10, or atleast 11, or at least 12, or at least 13, or at least 14, or at least15, or at least 16, or at least 17, or at least 18, or at least 19, orat least 20, or at least 21, or at least 22, or at least 23, or at least24, or at least 25, or at least 26, or at least 27, or at least 28, orat least 29, or at least 30, or at least 31, or at least 32, or at least33, or at least 34, or at least 35, or at least 36, or at least 37, orat least 38, or at least 39, or at least 40, or at least 41, or at least42, or at least 43, or at least 44, or at least 45, or at least 46, orat least 47, or at least 48, or at least 49, or at least 50, or at least51, or at least 52, or at least 53, or at least 54, or at least 55, orat least 56, or at least 57, or at least 58, or at least 59, or at least60, or at least 61, or at least 62, or at least 63, or at least 64, orat least 65, or at least 66, or at least 67, or at least 68, or at least69, or at least 70, or at least 71, or at least 72, or at least 73, orat least 74, or at least 75, or at least 76, or at least 77, or at least78, or at least 79, or at least 80, or at least 81, or at least 82, orat least 83, or at least 84, or at least 85, or at least 86, or at least87, or at least 88, or at least 89, or at least 90, or at least 91, orat least 92, or at least 93, or at least 94, or at least 95, or at least96, or at least 97, or at least 98, or at least 99, or at least 100contiguous nucleotides from any one of SEQ NOS: 1 or 3 wherein one ofthe nucleotides is located at the site of single nucleotide polymorphism(SNP) corresponding to single nucleotide polymorphism (SNP) at rs2230199on human chromosome 19 as set out herein or the complement thereof, andoptionally; wherein the isolated nucleic acid molecule has a maximumlength of 100 said contiguous nucleotides, or a maximum length of 90said contiguous nucleotides, or a maximum length of 80 said contiguousnucleotides, or a maximum length of 70 said contiguous nucleotides, or amaximum length of 60 said contiguous nucleotides, or a maximum length of50 said contiguous nucleotides, or a maximum length of 40 saidcontiguous nucleotides, or a maximum length of 30 said contiguousnucleotides, or a maximum length of 20 said contiguous nucleotides.

Oligonucleotides can be linked to a second moiety, which can be anothernucleic acid molecule to provide, for example, a tail sequence (e.g., apolyadenosine tail), an adapter sequence (e.g., phage M13 universal tailsequence), etc. Alternatively, the moiety might be one that facilitateslinkage to a solid support or a detectable label, e.g., a radioactivelabel, a fluorescent label, a chemiluminescent label, a paramagneticlabel, etc.

Nucleic acid sequences shown in SEQ ID NO: 1, 3 or 4, or fragmentsthereof, may be used for diagnostic purposes for detection ofpolypeptide expression.

DNA encoding a polypeptide can also be used in the diagnosis of agerelated macular degeneration. For example, the nucleic acid sequence canbe used in hybridization assays of biopsies or autopsies to polymorphicvariants associated with increased risk of AMD (e.g., Southern orNorthern blot analysis, in situ hybridization assays).

Expression of a polypeptide during embryonic development can also bedetermined using nucleic acid encoding the polypeptide, particularlyproduction of a functionally impaired polypeptide that is the cause ofage related macular degeneration. In situ hybridizations using apolypeptide as a probe can be employed to predict problems related toage related macular degeneration.

Included as part of this invention are nucleic acid vectors, oftenexpression vectors, which contain a nucleotide sequence set forth in theSEQ ID NO:1 or 3, or a fragment thereof. A vector is a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked and can include a plasmid, cosmid, or viral vector. Thevector can be capable of autonomous replication or it can integrate intoa host DNA. Viral vectors may include replication defectiveretroviruses, adenoviruses and adeno-associated viruses for example.

A vector can include a nucleotide sequence from SEQ ID NO: 1 or 3 or afragment thereof, in a form suitable for expression of an encodedprotein or nucleic acid in a host cell. The recombinant expressionvector generally includes one or more regulatory sequences operativelylinked to the nucleic acid sequence to be expressed. A regulatorysequence includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Regulatory sequences includethose that direct constitutive expression of a nucleotide sequence, aswell as tissue-specific regulatory and/or inducible sequences. Thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression ofpolypeptide desired, etc. Expression vectors can be introduced into hostcells to produce the desired polypeptides, including fusionpolypeptides.

Recombinant expression vectors can be designed for expression ofpolypeptides in prokaryotic or eukaryotic cells. For example, thepolypeptides can be expressed in E. coli, insect cells (e.g., usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further by Goeddel (31).A recombinantexpression vector can also be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

Expression of polypeptides in prokaryotes can be carried out in E. coliwith vectors containing constitutive or inducible promoters directingthe expression of either fusion or non-fusion polypeptides. Fusionvectors add a number of amino acids to a polypeptide. Such fusionvectors typically serve to increase expression of recombinantpolypeptide, to increase the solubility of the recombinant polypeptideand/or to aid in the purification of the recombinant polypeptide byacting as a ligand during purification. Often, a proteolytic cleavagesite is introduced at the junction of the fusion moiety and therecombinant polypeptide to enable separation of the recombinantpolypeptide from the fusion moiety after purification of the fusionpolypeptide. Such enzymes, and their cognate recognition sequences,include Factor Xa, thrombin and enterokinase. Typical fusion expressionvectors include pGEX (Pharmacia Biotech Inc;), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding polypeptide, orpolypeptide A, respectively, to the target recombinant polypeptide.

Purified fusion polypeptides can be used in screening assays and togenerate antibodies specific for polypeptides.

Expressing a polypeptide in host bacteria with an impaired capacity toproteolytically cleave the recombinant polypeptide can be used tomaximize recombinant polypeptide expression (32). The nucleotidesequence of the nucleic acid to be inserted into an expression vectorcan be changed so that the individual codons for each amino acid arethose preferentially utilized in E. coli (33).

When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. Recombinant mammalian expression vectors can becapable of directing expression of the nucleic acid in a particular celltype (e.g., tissue-specific regulatory elements are used to express thenucleic acid). Examples of suitable tissue-specific promoters include analbumin promoter(34), lymphoid-specific promoters (35) (36), promotersof immunoglobulins(37;38), neuron-specific promoters (39),pancreas-specific promoters (40), and mammary gland-specific promoters(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and EuropeanApplication Publication No. 264,166). Developmentally-regulatedpromoters are sometimes utilized, for example, the murine hoxpromoters(41) and the .alpha.-fetopolypeptide promoter(42).

Vectors can be introduced into host cells via conventionaltransformation or transfection techniques. The terms transformation andtransfection refer to a variety of techniques known for introducingforeign nucleic acid (e.g., DNA) into a host cell, including calciumphosphate or calcium chloride co-precipitation, transduction/infection,DEAE-dextran-mediated transfection, lipofection, or electroporation.

A host cell can be used to produce a polypeptide. Accordingly, methodsfor producing a polypeptide using the host cells are included as part ofthis invention. Such a method can include culturing host cells intowhich a recombinant expression vector encoding a polypeptide has beenintroduced in a suitable medium such that the polypeptide is produced.The method can further include isolating the polypeptide from the mediumor the host cell.

Polypeptides can be expressed in transgenic animals or plants byintroducing a nucleic acid encoding the polypeptide into the genome ofan animal. In certain embodiments the nucleic acid is placed under thecontrol of a tissue specific promoter, e.g., a milk or egg specificpromoter, and recovered from the milk or eggs produced by the animal.Also included is a population of cells from a transgenic animal.

Isolated polypeptides encoded by a nucleotide sequence from SEQ ID NO: 1or 3, or a fragment thereof, can be synthesized. Isolated polypeptidesinclude both the full-length polypeptide and the mature polypeptide(i.e., the polypeptide minus the signal sequence or propeptide domain).An isolated, or purified, polypeptide or protein is substantially freeof cellular material or other contaminating proteins from the cell ortissue source from which the protein is derived, or is substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized. Substantially free means a preparation of a polypeptidehaving less than about 5% (by dry weight) of contaminating protein, orof chemical precursors or non-target chemicals. When the desiredpolypeptide is recombinantly produced, it is typically substantiallyfree of culture medium, specifically, where culture medium representsless than about 10% of the polypeptide preparation.

Also, polypeptides may exist as chimeric or fusion polypeptides. As usedherein, a “target chimeric polypeptide” or “target fusion polypeptide”includes a target polypeptide linked to a different polypeptide. Thetarget polypeptide in the fusion polypeptide can correspond to an entireor nearly entire polypeptide as it exists in nature or a fragmentthereof. The other polypeptide can be fused to the N-terminus orC-terminus of the target polypeptide.

Fusion polypeptides can include a moiety having high affinity for aligand. For example, the fusion polypeptide can be a GST-target fusionpolypeptide in which the target sequences are fused to the C-terminus ofthe GST sequences, or a polyhistidine-target fusion polypeptide in whichthe target polypeptide is fused at the N- or C-terminus to a string ofhistidine residues. Such fusion polypeptides can facilitate purificationof recombinant target polypeptide. Expression vectors are commerciallyavailable that already encode a fusion moiety (e.g., a GST polypeptide),and a nucleotide sequence from SEQ ID NO: 1, 3 or 4, or a fragmentthereof, or a substantially identical nucleotide sequence thereof, canbe cloned into an expression vector such that the fusion moiety islinked in-frame to the target polypeptide. Further, the fusionpolypeptide can be a target polypeptide containing a heterologous signalsequence at its N-terminus. In certain host cells (e.g., mammalian hostcells), expression, secretion, cellular internalization, and cellularlocalization of a target polypeptide can be increased through use of aheterologous signal sequence. Fusion polypeptides can also include allor a part of a serum polypeptide (e.g., an IgG constant region or humanserum albumin).

Target polypeptides can be used as immunogens to produce anti-targetantibodies in a subject, to purify the polypeptide ligands or bindingpartners.

Polypeptides can be differentially modified during or after translation,e.g., by glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to an antibody molecule or other cellular ligand, etc.Any known modification including specific chemical cleavage by cyanogenbromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄; acetylation,formylation, oxidation, reduction; metabolic synthesis in the presenceof tunicamycin; etc. may be used. Additional post-translationalmodifications include, for example, N-linked or O-linked carbohydratechains, processing of N-terminal or C-terminal ends), attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression. The polypeptide fragments may also be modified with adetectable label, such as an enzymatic, fluorescent, isotopic oraffinity label to allow for detection and isolation of the polypeptide.

Pharmacogenomics is a discipline that involves tailoring a treatment fora subject according to the subject's genotype. For example, based uponthe outcome of a prognostic test, a clinician or physician may targetpertinent information and preventative or therapeutic treatments to asubject who would be benefited by the information or treatment and avoiddirecting such information and treatments to a subject who would not bebenefited (e.g., the treatment has no therapeutic effect and/or thesubject experiences adverse side effects). As therapeutic approaches forage related macular degeneration continue to evolve and improve, thegoal of treatments for age related macular degeneration relateddisorders is to intervene even before clinical signs manifestthemselves. Thus, genetic markers associated with susceptibility to agerelated macular degeneration prove useful for early diagnosis,prevention and treatment of age related macular degeneration.

The following is an example of a pharmacogenomic embodiment. Aparticular treatment regimen can exert a differential effect dependingupon the subject's genotype. Where a candidate therapeutic exhibits asignificant beneficial interaction with a prevalent allele and acomparatively weak interaction with an uncommon allele (e.g., an orderof magnitude or greater difference in the interaction), such atherapeutic typically would not be administered to a subject genotypedas being homozygous for the uncommon allele, and sometimes notadministered to a subject genotyped as being heterozygous for theuncommon allele. In another example, where a candidate therapeutic isnot significantly toxic when administered to subjects who are homozygousfor a prevalent allele but is comparatively toxic when administered tosubjects heterozygous or homozygous for an uncommon allele, thecandidate therapeutic is not typically administered to subjects who aregenotyped as being heterozygous or homozygous with respect to theuncommon allele.

Methods of the invention are applicable to pharmacogenomic methods fordetecting, preventing, alleviating and/or treating age related maculardegeneration. For example, a nucleic acid sample from an individual maybe subjected to a genetic test. Where one or more polymorphic variantsassociated with increased risk of age related macular degeneration areidentified at SNPs in the individual, information for detecting,preventing or treating age related macular degeneration and/or one ormore age related macular degeneration detection, prevention and/ortreatment regimens then may be directed to and/or prescribed to thatindividual.

In certain embodiments, a detection, preventative and/or treatmentregimen is specifically prescribed and/or administered to individualswho will most benefit from it based upon their risk of developing agerelated macular degeneration assessed by the methods described herein.Methods are thus provided for identifying a subject at risk of agerelated macular degeneration and then prescribing a detection,therapeutic or preventative regimen to individuals identified as beingat increased risk of age related macular degeneration. Thus, certainembodiments are directed to methods for treating age related maculardegeneration in a subject, reducing risk of age related maculardegeneration in a subject, or early detection of age related maculardegeneration in a subject, which comprise: detecting the presence orabsence of a polymorphic variant associated with age related maculardegeneration at a SNP in a nucleotide sequence set forth in SEQ ID NOs:1and 3, and prescribing or administering an age related maculardegeneration treatment regimen, preventative regimen and/or detectionregimen to a subject from whom the sample originated where the presenceof polymorphic variants associated with age related macular degenerationare detected at one or more SNPs in the nucleotide sequence. In thesemethods, genetic results may be utilized in combination with other testresults to diagnose age related macular degeneration as described above.

The use of certain age related macular degeneration treatments are knownin the art, and include surgery, chemotherapy and/or radiation therapy.Any of the treatments may be used in combination to treat or prevent agerelated macular degeneration (e.g., surgery followed by radiationtherapy or chemotherapy).

Pharmacogenomics methods also may be used to analyze and predict aresponse to a age related macular degeneration treatment or a drug. Forexample, if pharmacogenomics analysis indicates a likelihood that anindividual will respond positively to a age related macular degenerationtreatment with a particular drug, the drug may be administered to theindividual.

Conversely, if the analysis indicates that an individual is likely torespond negatively to treatment with a particular drug, an alternativecourse of treatment may be prescribed. A negative response may bedefined as either the absence of an efficacious response or the presenceof toxic side effects. The response to a therapeutic treatment can bepredicted in a background study in which subjects in any of thefollowing populations are genotyped: a population that respondsfavorably to a treatment regimen, a population that does not respondsignificantly to a treatment regimen, and a population that respondsadversely to a treatment regiment (e.g., exhibits one or more sideeffects). These populations are provided as examples and otherpopulations and subpopulations may be analyzed. Based upon the resultsof these analyses, a subject is genotyped to predict whether he or shewill respond favorably to a treatment regimen, not respond significantlyto a treatment regimen, or respond adversely to a treatment regimen.

The methods described herein also are applicable to clinical drugtrials. Polymorphic variants indicative of response to an agent fortreating age related macular degeneration or to side effects to an agentfor treating age related macular degeneration may be identified at oneor more SNPs. Thereafter, potential participants in clinical trials ofsuch an agent may be screened to identify those individuals most likelyto respond favorably to the drug and exclude those likely to experienceside effects. In that way, the effectiveness of drug treatment may bemeasured in individuals who respond positively to the drug, withoutlowering the measurement as a result of the inclusion of individuals whoare unlikely to respond positively in the study and without riskingundesirable safety problems.

Thus, another embodiment is a method of selecting an individual forinclusion in a clinical trial of a treatment or drug comprising thesteps of: (a) obtaining a nucleic acid sample from an individual; (b)determining the identity of a polymorphic variant which is associatedwith a positive response to the treatment or the drug, or a polymorphicvariant which is associated with a negative response to the treatment orthe drug at at least one SNP in the nucleic acid sample, and (c)including the individual in the clinical trial if the nucleic acidsample contains the polymorphic variant associated with a positiveresponse to the treatment or the drug or if the nucleic acid samplelacks said polymorphic variant associated with a negative response tothe treatment or the drug. The SNP may be in a sequence selectedindividually or in any combination from the C3 genomic sequencedisclosed in the table. Step (c) can also include administering the drugor the treatment to the individual if the nucleic acid sample containsthe polymorphic variant associated with a positive response to thetreatment or the drug and the nucleic acid sample lacks the polymorphicvariant associated with a negative response to the treatment or thedrug.

A peptide nucleic acid, or PNA, refers to a nucleic acid mimic such as aDNA mimic, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of a PNA can allow for specifichybridization to DNA and RNA under conditions of low ionic strength.Synthesis of PNA oligomers can be performed using standard solid phasepeptide synthesis protocols as described, for example, in Hyrup et al.(71), and Perry-O'Keefe et al. (70).

PNA nucleic acids can be used in prognostic and diagnostic applications.For example, PNAs can be used in the analysis of SNPs in a gene, (e.g.,by PNA-directed PCR clamping); as artificial restriction enzymes whenused in combination with other enzymes, (e.g., S1 nucleases(71) or asprobes or primers for DNA sequencing or hybridization (71;72).

A further aspect of the invention provides an antibody molecule thatbinds specifically to a vairiant C3 polypeptide i.e. a polypeptideencoded by a nucleotide sequence comprising polymorphic variants at oneor more SNPs described herein. For example, an antibody molecule maybind specifically to the C3F polypeptide which comprises an R102Gsubstitution which is encoded by the coding sequence comprising the Gallele of SNP rs2230199. Such an antibody binds preferentially to theC3F polypeptide relative to C3S polypeptide which lacks the R102Gsubstitution.

A method of identifying and/or obtaining an antibody specific for C3Fpolypeptide may comprise;

-   -   providing a population of antibody molecules specific for C3F        polypeptide,    -   contacting said population with a C3S polypeptide,    -   identifying one or more members of said population which bind        preferentially to C3F relative to C3S polypeptide.

Antibody molecules may be useful both in the diagnosis of AMD, inaccordance with the invention.

Antibodies that are specific for a C3 polypeptide may be obtained usingtechniques that are standard in the art. An immunogen typically is usedto prepare antibodies by immunizing a suitable subject, (e.g., rabbit,goat, mouse or other mammal). An appropriate immunogenic preparation cancontain, for example, recombinantly expressed chemically synthesizedpolypeptide. The preparation can further include an adjuvant, such asFreund's complete or incomplete adjuvant, or a similar immunostimulatoryagent.

Amino acid polymorphisms can be detected using antibodies specific forthe altered epitope by western analysis after the electrophoresis ofdenatured proteins. Protein polymorphism can also be detected usingfluorescently identified antibodies which bind to specific polymorphicepitopes and detected in whole cells using fluorescence activated cellsorting techniques (FACS). Polymorphic protein sequence may also bedetermined by NMR spectroscopy or by x-ray diffraction studies. Further,determination of polymorphic sites in proteins may be accomplished byobserving differential cleavage by specific or non specific proteases.

An antibody is an immunoglobulin molecule or immunologically activeportion thereof, i.e., an antigen-binding portion. Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)₂ fragments which can be generated by treating theantibody with an enzyme such as pepsin. An antibody can be polyclonal,monoclonal, or recombinant (e.g., a chimeric or humanized), fully human,non-human (e.g., murine), or a single chain antibody.

A full-length polypeptide or antigenic peptide fragment encoded by atarget nucleotide sequence can be used as an immunogen or can be used toidentify antibodies made with other immunogens, e.g., cells, membranepreparations, and the like. An antigenic peptide often includes at least8 amino acid residues of the amino acid sequences encoded by anucleotide sequence of one of SEQ ID NOs:1 and 3, and encompasses anepitope. Antigenic peptides sometimes include 10 or more amino acids, 15or more amino acids, 20 or more amino acids, or 30 or more amino acids.Hydrophilic and hydrophobic fragments of polypeptides sometimes are usedas immunogens.

Epitopes encompassed by the antigenic peptide are regions located on thesurface of the polypeptide (e.g., hydrophilic regions) as well asregions with high antigenicity. For example, an Emini surfaceprobability analysis of the human polypeptide sequence can be used toindicate the regions that have a particularly high probability of beinglocalized to the surface of the polypeptide and are thus likely toconstitute surface residues useful for targeting antibody production.The antibody may bind an epitope on any domain or region on polypeptidesfor use in the invention.

An antibody (e.g., monoclonal antibody) can be used to isolate targetpolypeptides by standard techniques, such as affinity chromatography orimmunoprecipitation. Moreover, an antibody can be used to detect atarget polypeptide (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of thepolypeptide. Antibodies can be used diagnostically to monitorpolypeptide levels in tissue as part of a clinical testing procedure,e.g., to determine the efficacy of a given treatment regimen. Detectioncan be facilitated by coupling (i.e., physically linking) the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H. Also, an antibody can be utilized as a test molecule for determiningwhether it can treat age related macular degeneration , and as atherapeutic for administration to a subject for treating age relatedmacular degeneration.

An antibody can be made by immunizing with a purified antigen, or afragment thereof, a membrane associated antigen, tissues, e.g., crudetissue preparations, whole cells, preferably living cells, lysed cells,or cell fractions.

Included as part of this invention are antibodies which bind only anative polypeptide, only denatured or otherwise non-native polypeptide,or which bind both, as well as those having linear or conformationalepitopes. Conformational epitopes sometimes can be identified byselecting antibodies that bind to native but not denatured polypeptide.Also featured are antibodies that specifically bind to a polypeptidevariant associated with age related macular degeneration.

Preferably, an antibody displays increased binding to the C3Fpolypeptide relative to the C3S polypeptide.

The examples set forth below are intended to illustrate but not limitthe invention.

Age-related macular degeneration is the most common cause of blindnessin Western populations. Susceptibility is influenced by age and bygenetic and environmental factors.

Complement activation is implicated in the pathogenesis. We tested foran association between age-related macular degeneration and 13 singlenucleotide polymorphisms (SNPs) spanning the complement genes C3 and C5in case subjects and control subjects from the southeastern region ofEngland. All subjects were examined by an ophthalmologist and hadindependent grading of fundus photographs to confirm their diseasestatus. To test for replication of the most significant findings, wegenotyped a set of Scottish cases and controls. The common functionalpolymorphism rs2230199 (Arg102Gly) in the C3 gene, corresponding to theelectrophoretic variants C3S (slow) and C3F (fast), was stronglyassociated with age-related macular degeneration in both the Englishgroup (603 cases and 350 controls, P=5.9×10-5) and the Scottish group(244 cases and 351 controls, P=5.0×10-5). The odds ratio for age-relatedmacular degeneration in C3 S/F heterozygotes as compared with S/Shomozygotes was 1.7 (95% confidence interval [CI], 1.3 to 2.1); for F/Fhomozygotes, the odds ratio was 2.6 (95% CI, 1.6 to 4.1). The estimatedpopulation attributable risk for C3F was 22%. Complement C3 is importantin the pathogenesis of age-related macular degeneration. This findingfurther underscores the influence of the complement pathway in thepathogenesis of this disease.

The inventors of the present invention have discovered a single basepair polymorphism that is present in a highly significant percentage ofthe genetic DNA of individuals affected with age related maculardegeneration while only present in a smaller percentage of individualswho are not known to be affected by the disease.

For individuals with age-related macular degeneration, the distributionof polymorphic alleles at position 6669387 of chromosome 19, foundwithin the C3 gene, was different from those without age-related maculardegeneration (Table 1). The trend test for risk associated with carryingthe C allele (on the positive reference strand of the human genome) hadan empirical p-value of 0.000059225, and the correspondingMantel-Haenszel odds ratio for trend is 1.600 (Table 1). These datafurther suggest that this marker, located within the C3 gene, isassociated with age-related macular degeneration risk and that the Callele at position 6669387 of chromosome 19 is associated with anincreased risk of developing age-related macular degeneration. The Callele at position 6669387 of the positive strand corresponds to the Gallele within the negative strand, in which is found the coding sequencefor C3.

TABLE 1 rs no. 2230199 Chromosome; Position 19; 6669387 Gene Name C3 SEQID NO; Position 3; 2274 Genotype; Phenotype n = C; increased risk(positive strand relative to the human reference sequence version 36.1)Hardy-Weinberg 0.86594 Case Allele Odds Flag B AA AB BB Model p-ValueRatio 0 C 223 109 14 Trend 0.000059 1.600 1 C 303 242 45

The present invention has been described in detail by way ofillustration and example in order to acquaint others skilled in the artwith the invention, its principles and its practical application.Particular formulations and processes of the present invention are notlimited to the descriptions of the specific embodiments presented, butrather the descriptions and examples should be viewed in terms of theclaims that follow and their equivalents. While some of the examples anddescriptions above include some conclusions about the way the inventionmay function, the inventors do not intend to be bound by thoseconclusions and functions, but put them forth only as possibleexplanations.

It is to be further understood that the specific embodiments of thepresent invention as set forth are not intended as being exhaustive orlimiting of the invention, and that many alternatives, modifications andvariations will be apparent to those of ordinary skill in the art inlight of the foregoing examples and detailed description. Accordingly,this invention is intended to embrace all such alternatives,modifications and variations that fall within the spirit and scope ofthe following claims.

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1-29. (canceled)
 30. A method for assessing the probability of atherapeutic; or toxic response to a chemical, protein, nucleicacid-based therapeutic or other biological, naturally or syntheticentity in a human subject who has or who may have age-related maculardegeneration or any pathological process substantially similar, themethod comprising the steps of: i) obtaining a biological sample from ahuman subject; ii) analyzing the sample to determine whether the subjectcarries one or more of: a) C or G at rs2230199 of the complement 3 (C3)gene, or b) the amino acid arginine or glycine at position 102 of the C3protein.
 31. A method according to claim 30 wherein the biologicalsample is analyzed by a method comprising assaying DNA in said samplefor the presence of at least one allele of the complement component 3(C3) gene comprising a G nucleotide at the single nucleotidepolymorphism (SNP) rs2230199 at the position corresponding to position366 of SEQ ID NO:1, wherein the presence of at least one allele of theC3 gene comprising a G nucleotide at the single nucleotide polymorphism(SNP) rs2230199 at the position corresponding to position 366 of SEQ IDNO:1 is indicative of an increased probability of a therapeutic or toxicresponse to a chemical, protein, nucleic acid-based therapeutic or otherbiological, naturally or synthetic entity in the human subject.
 32. Amethod of claim 31 wherein said assaying comprises; amplifying the DNAin the presence of a pair of primers wherein a first of the primerscomprises at least 10 consecutive nucleotides selected from the sequenceidentified as SEQ ID NO 1 and located upstream of the base located atposition 366 of the sequence and a second primer comprising at least 10consecutive nucleotides selected from within the same sequence andlocated downstream of the base located at position 366; and determiningthe identity of the base in the amplified genetic material thatcorresponds to position
 366. 33. A method according to claim 32 whereinthe amplified genetic material is between about 16 and about 2000nucleotides in length,
 34. A method according to claim 31 wherein saidassaying comprises; contacting the biological sample with a reagentwhich specifically hybridizes under stringent hybridization conditionsto an allele of the complement component 3 (C3) gene comprising a Gnucleotide at the single nucleotide polymorphism (SNP) rs2230199 at theposition corresponding to position 366 of SEQ ID NO:1, or a reagentwhich specifically hybridizes under stringent hybridization conditionsto an allele of the complement component 3 (C3) gene comprising a Cnucleotide at the single nucleotide polymorphism (SNP) rs2230199 at theposition corresponding to position 366 of SEQ ID NO:1, and detecting theformation of a hybridized duplex.
 35. A method according to claim 34 inwhich detection is carried out by a process which may be selected fromthe group consisting of: allele-specific probe hybridization,allele-specific primer extension, allele-specific amplification,sequencing, 5′ nuclease digestion, molecular beacon assay,oligonucleotide ligation assay, size analysis, and single-strandedconformation polymorphism.
 36. A method according to claim 34 whereinthe reagent is a polynucleotide probe.
 37. A method according to claim36 wherein the polynucleotide probe is contained in a rnicroarray.
 38. Amethod according to claim 30 wherein the biological sample is analyzedby a method comprising assaying said sample for the presence of a C3protein comprising a glycine amino acid at the position corresponding toposition 102 of SEQ ID NO:2; wherein the presence of a C3 proteincomprising a glycine amino acid at the position corresponding toposition 102 of SEQ ID N0:2 is indicative of an increased probability ofa therapeutic; or toxic response to a chemical, protein, nucleicacid-based therapeutic; or other biological, naturally or synthetic;entity in the human subject.
 39. A method according to claim 38 whereinsaid assaying comprises; contacting a test sample with a specificbinding member which binds to a variant polypeptide encoded by anucleotide sequence which comprises the nucleotide sequence of SEQ IDNO:1 or SEQ ID NO: 3 with a polymorphic variant sequence at a site ofsingle nucleotide polymorphism (SNP) therein, and detecting the bindingof the specific binding member to polypeptide in the sample.
 40. Amethod according to claim 30, wherein the subject is asymptomatic ofmacular degeneration.
 41. A method according to claim 30, wherein thesubject has been diagnosed as having symptoms of macular degeneration.42. A method according to claim 30, wherein the sample is from blood,saliva, sputum, urine, cell scrapings or biopsy tissue.
 43. The methodof claim 31 further comprising determining whether the subject ishomozygous or heterozygous for said polymorphism.
 44. A methodcomprising the steps of: assaying a biological sample from a human anddetecting the presence of (i) at least one allele of the complementcomponent 3 (C3) gene comprising a G nucleotide at the single nucleotidepolymorphism (SNP) rs2230199 at the position corresponding to position366 of SEQ ID NO: 1; or (ii) a C3 protein comprising a glycine aminoacid at the position corresponding to position 102 of SEQ ID NO: 2;wherein the presence of at least one allele of the C3 gene comprising anucleotide at the single nucleotide polymorphism (SNP) rs2230199 at theposition corresponding to position 366 of SEQ ID NO: 1 or a C3 proteincomprising a glycine amino add at the position corresponding to position102 of SEQ ID NO:2 is indicative of an increased risk of development ofmacular degeneration in said human.
 45. The method of claim 44, whereinsaid biological sample is DNA and said assaying comprises: amplifyingthe DNA in the presence of a pair of primers wherein a first primercomprises at least 10 consecutive nucleotides of one of SEQ ID NO: 1 orthe complement of SEQ ID NO:1 and is located upstream of the baselocated at position 366 of SEQ ID NO:1, and a second primer comprises atleast 10 consecutive nucleotides of the other of SEQ ID NO:1 or thecomplement of SEQ ID NO:1 and is located downstream of the base locatedat position 366 of SEQ ID NO:1; and determining the identity of the basein the amplified genetic material that corresponds to position 366 ofSEQ ID NO:
 1. 46. A method comprising the steps of: assaying abiological sample from a human and detecting the presence of (i) atleast one allele of the complement component 3 (C3) gene comprising a Gnucleotide at the single nucleotide polymorphism (SNP) rs2230199 at theposition corresponding to position 366 of SEQ ID NO: 1; or (ii) a C3protein comprising a glycine amino acid at the position corresponding toposition 102 of SEQ ID NO: 2; wherein the presence of at least oneallele of the C3 gene comprising a nucleotide at the single nucleotidepolymorphism (SNP) rs2230199 at the position corresponding to position366 of SEQ ID NO: 1 or a C3 protein comprising a glycine amino add atthe position corresponding to position 102 of SEQ ID NO:2 is indicativeof an increased risk of development of macular degenerationcharacterized by geographic atrophy or exudative disease in said human.47. The method of claim 44, wherein said biological sample is DNA andsaid assaying comprises: amplifying the DNA in the presence of a pair ofprimers wherein a first primer comprises at least 10 consecutivenucleotides of one of SEQ ID NO: 1 or the complement of SEG ID Nal andis located upstream of the base located at position 366 of SEQ ID NO:1,and a second primer comprises at least 10 consecutive nucleotides of theother of SEQ ID NO:1 or the complement of SEC) ID NO:1 and is locateddownstream of the base located at position 366 of SEQ ID NO:1; anddetermining the identity of the base in the amplified genetic materialthat corresponds to position 366 of SEQ ID NO: 1.